ENZYMATIC-SYNTHESIS OF NAD(+) WITH THE SPECIFIC INCORPORATION OF ATOMIC LABELS

An enzymatic synthesis is described for the production of NAD(+) label ed with a radioactive or stable isotope at any desired position in the AMP or NMN(+) portions of the molecule. In the first step, ten enzyme -catalyzed reactions are coupled for the synthesis of nicotinic acid a denine dinucleotide (NaAD(+)) from glucose, nicotinic acid, and ATP. N AD(+) is formed from NaAD(+) and glutamine in the second step. Oxidize d nicotinamide adenine dinucleotide was synthesized with H-3, C-14, or N-15 label specifically incorporated in the ribose or nicotinamide of the NMN(+) portion of NAD(+) as [H(N)1'H-3]NAD(+), [H(N)2'-H-3]NAD(+) , [H(N)4'-H-3]NAD(+), [H(N)5'-H-3]NAD(+), [C(N)1'-C-14]NAD(+), [C(N)5' -C-14]NAD(+), [N(N)1-N-15, C(N)1'-C-14]NAD(+), and [N(N)1-N-15, C(N)5' -C-14]NAD(+). Nuclear magnetic resonance spectroscopy of [H(N)2'-H-2]N AD(+) as well as enzymatic degradation were used to verify the positio n of labels. Appropriately labeled glucose, ribose 5-phosphate, or nic otinic acid were the starting materials and were converted to NAD(+) u sing enzymes from the pentose pathway and the pathway for NAD(+) de no vo synthesis. Yields of purified NAD(+) to 96% were obtained from star ting glucose. The labeled NAD(+) is catalytically competent and is chr omatographically and spectrophotometrically indistinguishable from aut hentic NAD(+). By using specifically labeled ATP as a precursor (Parki n, D. W.; Schramm, V. L. Biochemistry 1987, 26, 913-920), the method i s readily adaptable for the synthesis of NAD(+) with single or multipl e atomic labels at various positions in the AMP portion of the molecul e. NAD(+) was synthesized from [8-C-14]ATP to give [C(A)8-C-14]NAD(+) as an example. Together these methods provide a general scheme for the efficient synthesis of NAD(+) of high purity with H-3, C-14, Or Other labels at any nonexchangeable position of the NMN(+) or AMP portions of the NAD(+) molecule.